JPH03290193A - Physiologically active substance tan-1323, its production and use - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (R is group of formula II or formula III). USE:A vascularization inhibitor useful for the prevention and remedy of tumor. PREPARATION:A microbial strain belonging to genus Streptomyces [e.g. Streptomyces sp. S-45628 (FERM BP-2728)] is cultured at 24-28 deg.C and pH6-7 for 24-144hr preferably under aeration and agitation.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a physiologically active substance TAN1323 effective in the prevention and treatment of various diseases accompanied by abnormal proliferation of angiogenesis, a method for producing the same, and uses thereof.

Conventional technology Angiogenesis occurs during embryonic development, ovulation or placentation during the female sexual cycle, normal physiological conditions of humans or animals, wound healing, repair processes such as inflammation, and rapid proliferation of capillaries.
It is known to occur in many pathological conditions that increase and cause severe damage to tissues. Diseases caused by such a pathological increase in capillaries include diabetic retinopathy, retrolental fibroplasia, neovascularization associated with corneal transplantation, endometriosis, ocular tumors, and trachoma in the ophthalmology field, and in the dermatology field. Psoriasis and pyogenic granulomas, angioplasty and fibrous hemangioma in the pediatric field, hypertrophic scars and granulomas in the surgical field, rheumatoid arthritis and edematous sclerosis in the internal medicine field, etc. Diseases such as atherosclerosis and various tumors are known.

In particular, increased abnormal angiogenesis in diabetic retinopathy and trachoma can lead to blindness in many people, and
Rheumatoid arthritis causes abnormal angiogenesis in the joints or destruction of the cartilage in the joints, which causes problems for many people. Therefore, it is desired to develop a compound useful as a therapeutic or prophylactic drug for diseases accompanied by such abnormal growth of angiogenesis.

In addition, the rapid growth of tumors is thought to be due to the formation of new blood vessels induced by angiogenic factors produced by tumor cells, and angiogenesis inhibitors are expected to become new therapeutic drugs for various tumors. Exploratory research into inhibitors has begun [Jay Volkman (J, F.
olkman), Advances in Cancer Research
er Re5erach), 43 ] 75.
1985 George Kl.
ein) and Sitney Winehouse (S 1dne)
y Weinhouse)].

It is known that angiogenesis is inhibited by the combination of heparin or heparin fragments and so-called angiogenesis-inhibiting steroids (angiosLatic 5teroids), including coachrin [/A. Volkmann et al. , Folkma
net al, ), Science
, 221719 (1983); J. Volkman et al.

Folkman et al.), Ennals et al.
Annals of S
Urgery), 206374 (1987).

Furthermore, acetincarphonic acid (L-azetidi
ne2-carboxylic acid) and cis-hydroxyproline
line) and kolaken synthesis inhibitors, and kola-
Ken Florin Hydrokinrase (college)
n proline hydroxylase) inhibitors, collagen crosslink formation inhibitors, and β-cycloC dextrin tetradeca sulfate (β-cyclodext
It has been reported that combination use with rin-tetradecasulfate (Rin-tetradecasulfate) or heparin exhibits an angiogenesis inhibitory effect [T. Ingber and N.E.
Folkman, Laboratory Investigation
ation), 59 44 (+988)].

It has also been pointed out that the basement membrane and collagen synthesis in the basement membrane play an important role in angiogenesis [
M. E. Maragoutakis, M. Salmonica and M. Banautsakopoulos (M. E., Marag.
oudakis, M., Salmonika a.
ndM, P anoutsacopoulous)
, Sharnall 1 of 0 Huamakoronoo &.
Experimental Theraboutics (J,
Pharmacol, Exp.

Ther, ), 244 729 (1988), D. E. Ingher, H. H. Matry and N. E. Volkmann (D.
, J=4 A, Madri and J, F o
lkman), Endocrinolone (Endocri)
1] 9 1768 (1986
)].

Furthermore, fumagillin, produced by Aspergillus fumigatus and conventionally known as an antibacterial and antiprotozoal agent, exhibits a strong angiogenesis inhibitory effect, and heparin or β-cyclodextrin tetratheca It has been recognized that the combination with sulfate shows an even more effective angiogenesis inhibitory effect [Shea Volkman and T. Kanamaru (JF olk
Man and T, Kanamaru) et al., EP-
A325199].

In addition, the steroid hormone drug medroquine progesterone acetate (m
edroxyprogesterone aceta
It has been reported that it inhibits angiogenesis induced by various tumors in an angiogenesis evaluation system using te) or Usaki corneas [Brondenges of the
Academy of Science and Technology 9A (P
roceedings or the Nati
onalAcademy of 5science,
U.S.A.), 781176 (1981)]
. Also, indomethacin
), diclofena
c sodium), aspirin
Prostaglandin synthesis inhibitors such as Anticancer Re5ear
ch) 6 251 (1986)], mitoxantrone, bisantrene (bis
Anthracene-based anticancer drugs such as antrene) [Biochemical and Biophysical Research
Communication CB iochemical &
B 1 Physical Research C
communication), 140 901 (1
986)] has also been found to have angiogenesis inhibitory activity.

Also, recently, gold sodium thiomalate (gold sodium thiomalate)
), auranofin (auranofin), and other gold-containing anti-angiogenic drugs have been shown to exhibit angiogenesis-inhibiting activity, suggesting that at least a portion of the anti-rheumatic effects of these compounds are related to angiogenesis-inhibiting activity. [Journal of Clinical Investigation (Journal of Clinical Investigation)]
nicaC11nicalInvesti, 79
1440 (1987): Biochemical and Biophysical Research Communication (B i
chemical & biophysical
Research Communication) 1
54 205 (1988)].

In addition to the above-mentioned non-protein angiogenesis inhibitors, there are also some reports on protein-based angiogenesis inhibitors, such as
Cartilage-derived factors [Science,
193 70 (1976)], protamine (prota
mine) [8ichi + -(Nature),
297307 (19B2)], human retinal pigment epithelial cell-derived factor [Archives of Ophthalmology (Ar
ch, Ophthalmol, ), 103 18
70 (1985)], interferon [cancer
Research (Cancer Research),
47 5155 (1987)] are known, but
It is considered to have little practicality as a medicine.

7. Problems to be Solved by the Invention Although there are several reports as mentioned above regarding compounds that exhibit angiogenesis inhibitory activity, it is considered that their inhibitory activity is still insufficient for clinical application. Therefore, it is strongly desired to obtain a strong angiogenesis inhibitor of small molecules that can be used clinically.

Means for Solving the Problems In view of the above circumstances, the present inventors isolated and searched for a large number of microorganisms with the aim of finding new angiogenesis inhibitors with stronger inhibitory activity and fewer side effects, and found that certain We have discovered that microorganisms produce a novel angiogenesis inhibitor.

The microorganism belongs to the genus Streptomyces, and four active substances (TAN-1323A, B, and C) that strongly inhibit angiogenesis by culturing the microorganism in an appropriate medium.

D) was found to be able to accumulate in the bacterial cells. We isolated these active substances and confirmed from their physicochemical properties that two of the active substances (TAN-1323c and D) are new substances represented by the following formulas. Based on these findings, we further As a result of continued research, the present invention was completed.

That is, the present invention provides: (1) a novel physiologically active substance TAN-1323c and D or a salt thereof; (2) a physiologically active substance TAN-1323c and D that belongs to the genus Streptomyces;
-Culture microorganisms capable of producing 1,323c and/or D in a medium! 2. Physiologically active substance T in the culture
Physiologically active substance TAN characterized by producing and accumulating AN-1323C and/or D and collecting the same
-1323C and/or D, and (3) an angiogenesis inhibitor containing the physiologically active substance TAN-1323A, B, C and/or D or a salt thereof.

Physiologically active substances TAN-1323A and B of the present invention.

Bacteria that produce C and/or D (hereinafter sometimes simply referred to as TAN-1323) include Streptomyces (S t
Any microorganism that belongs to the genus Reptomyces and has the ability to produce TAN-1323 may be used.

An example of this is Streptomyces sp., which was isolated from soil collected from the coast of Miyazaki Prefecture.
reptomyces sp, )S-45628
strain (hereinafter sometimes abbreviated as rS-45628 strain)
can be mentioned.

Regarding the S-45628 strain, international
[International Journal
of Systematic Bacteriol
ogy), 16(3) 313-340 (1966)
] The properties examined based on the description are as follows. Note that the findings on the culture medium are as follows:
The cells were cultured at 28°C for 14 days and observed.

(1) Morphological characteristics The aerial hyphae of this strain extend in a simple branched manner from the substratum hyphae, which are well-branched, with an open spiral chain of spores (usually 10 or more) at the tip. Is it acceptable? Or, circular yarn is not accepted. The spores are oval in shape and the size is 0.6 x 0.
．． It is 8-0.9 μm and its surface is thorn-like.

(2) Growth status on various media Growth level (G) on various media, aerial hyphae (A M)
The coloring and tone, the color tone (R) of the back side, the presence or absence of soluble dye and its color tone (sp), etc. are as shown in Table 1. Regarding the description of colors, the standard color tone symbols shown in parentheses are Container Corboley/Yon Off America (Contaii+erCo
poration of America) Nosu 0 Color 3 Harmony 0 Manual (The Co
lor Harmony Manual) 4th edition, 19
According to 1958.

2 (3) Physiological properties a) Growth temperature range (yeast extract/malt extract agar medium,
Observation for 1 week) = 13-44°C b) Liquefaction of Seratin
, Negative 1) Hydrolysis of starch ・Positive process) Coagulation of skim milk, Negative Peptonization of skim milk ・Positive E) Formation of melanin-like pigment Tyrol agar Positive Peptone/Yeast extract/Iron agar: Positive (4) Carbon Assimilation of carbon sources (Prietnom Gottlieb agar medium) Assimilation of various carbon sources is shown in Table 2.

Table 2 12 Assimilate - Not assimilate (5) Cell wall composition 2,6-diaminopimelic acid in the whole cell hydrolyzate is L type.

From the above properties, it is clear that the S-45628 strain belongs to the genus Streptomyces.

This Streptomyces sp. 5 45628 strain was transferred to the Fermentation Research Institute on December 1, 1999.
The microorganism has been deposited with the deposit number IF0149B, 0 since the 3rd, and the microorganism has been deposited at the Microbial Technology Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan (FRY, 1-1-3 Tsukuba Futsuka, Ibaraki Prefecture, Japan). Since January 10, 1990, it has been deposited under the Budapest Treaty under the deposit number FERM Bl''271B.

The Streptomyces genus actinomycetes used in the method of the present invention are
Generally, its properties change easily, such as ultraviolet rays, X-rays,
TAN-1323, which is the subject of the present invention, can be easily mutated by artificial mutation methods using chemicals (e.g., nitrosoguanidine, ethyl methanesulfonic acid), etc. Anything that has production capacity can be used in the method of the present invention.

When culturing the bacteria producing TAN-1323, carbon sources such as glucose, maltose, lactose, blackstrap molasses, fats and oils (e.g., soybean oil, olive oil, etc.), organic acids (e.g., citric acid, succinic acid, etc.) are recommended. , gluconic acid, etc.) that can be assimilated by the producing bacteria are used as appropriate. Nitrogen sources include, for example, soybean flour, cottonseed flour, corn steep liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, etc. Compounds and inorganic nitrogen compounds can be used. In addition, as inorganic salts, for example, inorganic salts normally required for culturing actinomycetes, such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, potassium phosphate, and disodium phosphate, may be used alone or in appropriate combinations. be done. Also, T
sulfur compounds that can be assimilated by the bacteria that produce AN-1323,
For example, sulfates (e.g., ammonium sulfate, etc.), thiosulfates (e.g., ammonium thiosulfate, etc.), sulfites (e.g.,
Inorganic sulfur compounds such as ammonium sulfite), sulfur-containing amino acids (e.g. cystine, cysteine, L-thiazolidine-
4-carboxylic acid), hypotaurine, sulfur-containing peptides (e.g.
Addition of organic sulfur compounds such as glutathione or mixtures thereof to the culture medium may increase the production of the target product.

In addition, heavy metals such as ferrous sulfate and copper sulfate, and vitamin B
,, Vitamins such as biotin are also added as necessary. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether may be added to the medium. In addition, it helps the growth of producing bacteria, and TAN-
Organic or inorganic substances that promote the production of 1323 may be added as appropriate.

The culturing method may generally be carried out in the same manner as the production method of antibiotics, and solid culture or 1 flk culture may be used. In the case of liquid culture, any of static culture, agitation culture, shaking culture, aeration culture, etc. may be used, and aeration and agitation culture is particularly preferred. In addition, the culture temperature is about 15°C to 35°C.
°C range is preferred, more preferably about 24 °C to 28 °C, the pH of the medium is in the range of about 4 to 8, more preferably about 6 to 7, and for about 8 hours to 168 hours. , preferably about 24 hours to 14 hours
Incubate for 4 hours.

The produced physiological layer substance TAN71323 exists mainly in the bacterial cells rather than in the culture filtrate, so after separating the culture into the supernatant and the bacterial cells by centrifugation or filtration, the The bacterial cells can be extracted and purified using an organic solvent such as methanol, acetone, butanol, or ethyl acetate, or purified from an extract obtained by directly adding the above organic solvent to the culture. I can do it.

To collect TAN-1323 from the culture solution, since the substance is a neutral or acidic fat-soluble substance, separation and purification means commonly used for collecting such microbial metabolites are appropriately used.

For example, methods that utilize the difference in solubility with impurities, activated carbon, nonionic high porous resins, chromatography that uses various carriers such as carbon, alumina, and dextran gel, etc., are used alone or in combination. Ru.

To explain specifically how to collect TAN-1323 produced in a culture, first, the culture solution is filtered using a filter aid such as Hyfloth Parcel, and the resulting bacterial cells are treated with methanol or After stirring and extraction by adding an organic solvent capable of dissolving the compound such as acetone, the mixture is filtered using a filter aid. The extract is concentrated, pH neutralized and extracted with a water-immiscible organic solvent such as ethyl acetate.

Organic solvents used for extraction include ethyl acetate, esters such as butyl acetate, chloroporum, halokenated hydrocarbons such as methylene chloride, n-butanol,
Examples include alcohols such as 1-butanol.

After washing the extract with water, the crude powder obtained by condensation is subjected to silica gel column chromatography.

As a developing solvent, a combined solvent such as chloroform-methanol or hexane-ethyl acetate is used, and by increasing the proportion of polar solvent in each solvent system, separation from other impurities can be achieved. can be eluted.

For example, when using a mixed solvent of lyloform-methanol, compound TAN1323A is first added, then
TAN-1323B.

TAN-1323C and finally TAN-1323D are eluted in this order. Each component is isolated by concentrating the eluted fractions followed by crystallization from a solvent such as methanol-water. If the amount of production is small and there are many impurities, and purification is difficult, it may be possible to obtain crystals by repeating gel chromatography.

TAN-1323c and D thus isolated
The physicochemical properties of are as follows.

(]) TAN-1323C 1) Shape Colorless crystal 2) Melting point 167-1685°C 3) Specific rotation [α1 do 31.9° (C = 0.41D
MF) 4) Molecular formula・C458740,4 5) Elemental analysis value (as C45H7,○1.-1/2H20)・Calculated value: C,63,73,H,8,91 actual value・C,63,76H, 8,98 6) Ultraviolet absorption spectrum λ': gH (to E): 244 (490), 284 (2
30) nm 7) Infrared absorption spectrum (by KBr tablet method) as shown in FIG.

8) 'H nuclear magnetic resonance spectrum (300MHzd,...
- in DMSO) as shown in FIG.

9) 13C nuclear magnetic resonance spectrum (75MHzd6-
Chemical shift in DMSO, δppm) 164.0
(s), 142.1 (s), 141.6 (s)1
41.0(d), 132. , 9(d), 130.8
(d) 129.9(s), 129.0(d), 12
7.3(d) 126, 6(d), 122.4(d),
99.3(d).

95.4(d), 82.0(d), 78.2(d)
, 74.6(d), 74.3(d), 72.1(d)
, 72.1(d).

71.8(d), 71.0(d), 70.7(d)
, 69.3(d), 68.7(d), 58.6(
q), 55.1 (Q).

45.5(t), 43.7(d), 42.3(d), 4
1.0(d), 37.9(d), 36.7(t),
34.7(d).

34.6(d), 22.1(t), 21.9(q),
17.8(q), 17.4(q), 17.2(q)
, 16.2(q) 13.9(q), 13.5(q)
, 11.9(q), 9.8(q).

6.9(q) However, S: singlet, d: doublet, t triplet (tr
iplet), q, quartet.

10) Color reaction positive Niline molybdic acid, iodine, sulfuric acid negative, ninhydrin, Burton 11) Solubility Easily soluble: Ethyl acetate, methanol, dimethyl sulfoxide, dimethylformamide Hardly soluble: Hexane, water 12) Thin layer chromatography (carrier) , silica gel glass plate 60 F tsa, 0.25 mm, manufactured by Merck & Co., West Germany): 13) Acidic, neutral, basic: Neutral 14) Structural formula: % formula % () ) 4) Molecular formula: C43H66013 5) Elemental analysis value (as C4sHeeo1*・l/2H, O): Calculated value: C, 64, 56, H, 8, 44 Actual value: C, 64, 48, H, 8, 706) Ultraviolet absorption spectrum λ丑H (E Tomoe): 211 (375), 243 (560
), 284 (250) nm 7) Infrared absorption spectrum (by KBr tablet method): 3rd
As shown in the figure.

8) 1H nuclear magnetic resonance spectrum (300MHz, heavy DM
SO): As shown in Figure 4.

9) 13c nuclear magnetic resonance spectrum (75MHz, heavy DM
Chemical shift in SO, δppm): 165.6
(s), 164.2(s), 164.1(s).

142.2(s), 141.6(s), 141.1
(d)4 134, 7(d), 133.0(d), 132.6
(d).

130.3(d), 130.0(s), 129.2
(d).

127.8(d), 126.6(d), 122.5
(d) 99.3(s), 81.9(d), 78.2(d)
), 74.4(d), 74.4(d), 74.1(d)
, 72.1(d).

69.4(d), 58.6(q), 55.1(q), 4
5.6(t), 43.8(d), 42.1(d)
, 40.2(d).

38.0(t), 37.9(d), 34.7(d), 3
4.7(d), 22.2(t), 22.0(q),
17.4(q).

17.2(q), 16.2(q), 13.9(q)
, 13.0(q), 11.9(q), 9.8(q
), 6.9(q)10) Color reaction: Positive Nilinemolybdic acid, iodine, sulfuric acid Negative: Ninhydrin, Barton 11) Solubility: Easily soluble: Ethyl acetate, methanol, dimethyl sulfoxide, dimethylformamide Slightly soluble : hexane, water 12) Thin layer chromatography (carrier, silica gel glass plate 60 F, 0.25 mm, manufactured by Merck & Co., West Germany) 13) Acidic, neutral, basic ・Acidic 14) Structure Formula compounds TAN-1323C and D are derived from concanamycin, an 18-membered ring macrolide.
In addition, the co-produced TAN 1323A and B are each a new substance belonging to the concanamycin (co
ncanamycin) C and A
tics), 87 1333 (1984)].

Since compound TAN-1323D is an acidic substance, it can be treated with sodium salt, potassium salt, calcium salt, etc. using methods known per se.
Physiologically acceptable salts can be formed, such as metal salts such as rum salt, salts with organic amines such as ammonium salts or triethylamine salts.

TAN-1323 (A, BXC, and D) exhibits potent angiogenesis inhibitory activity and is extremely useful as a prophylactic and therapeutic agent for many of the diseases based on the abnormal proliferation of angiogenesis described above. In addition, antitumor activity based on strong angiogenesis inhibiting action can be expected.

The compound may be administered orally or parenterally to a mammal (e.g.
(1) tablets, granules, rabbits, monkeys and humans)
It is administered in the form of capsules, tablets, powders, injections, topical creams, or eye drops. Pharmaceutical compositions used with active substances by extrusion may contain suitable customary excipients (drug substances), e.g.
Excipients, binders, disintegrants, lubricants, colorants, flavoring agents, stabilizers, etc. may also be included. The composition also includes sustained release (susLa) using sustained release polymer 7mer.
It may also be administered using an ined release technique. For example, the composition may be made of ethylene-vinyl acetate copolymer pellets (ethylene vinyl acetate copolymer pellets).
cetatecopolymer pellet) and the pellet can be transplanted extrinsically into the tissue to be treated.

For example, when used for the treatment of tumors or diabetic retinopathy, a composition containing - with a pharmacologically acceptable carrier is administered orally or by intravenous injection.

More specifically, it depends on the patient's condition, the degree of disease,
For example, it is desirable to administer 50 μ9 to 50 mg per day orally or parenterally in 2 to 3 divided doses. TAN-1
No serious toxicity was observed for 323A, B, C or D at these doses. It is also used as eye drops for the treatment of retinopathy and trachoma, and can be instilled into the eye 1 to 4 times a day depending on the patient's condition.

Effect The angiogenesis inhibitory activity of the compound of the present invention can be demonstrated in the chorioallantoic membrane of 28 chicken embryos.
lantoic membrane.

CAM), Nei Volkman (JFolkm)
R, Crume et al.
T, al), Science,
2301375 (+985)] by a slightly modified method. That is, the eggshells of fertilized chicken eggs that had been cultured for 3 days were broken, the resulting chicken embryos were hung in a hammock shape using polyethylene in a plastic cup, and the culture was continued for an additional 6 days under aseptic conditions.

The test sample was prepared as follows. That is, a methanol solution of the compound of the present invention prepared aseptically and a 1% aqueous methyl cellulose solution are mixed in equal volumes, and the mixed solution 1 is prepared.
0μQ was gently dropped onto a polypropylene sheet and air-dried aseptically to form a sample-containing sample with a diameter of approximately 4++.
+m methylcellulose tissue is obtained. The methylcellulose TISF prepared in the above method was gently placed on the chicken embryo on the 6th day of culture after egg cleavage, and the chick embryo was continued to be cultured aseptically.24 and 48 hours later, around the TISF. The formed angiogenesis prevention saw 7 is viewed under a microscope (X
20SSMZ-10SNikon). Angiogenesis inhibitory activity (%) was calculated as the ratio of disks in which an angiogenesis inhibition zone was observed per the total number of tisphenes used in the test.

The angiogenesis inhibitory activity of the compounds of the present invention was measured by the shellless chicken embryo chorioallantoic membrane (CAM) method described above, and as shown in Table 3, it was revealed that the compounds had extremely strong angiogenesis inhibitory effects.

*Measurement value after 24 hours of cold application The present invention will be described in more detail below with reference to Examples. In addition, in the examples, unless otherwise specified, "%" means % by weight.
It is.

Example 1 2% glucose, 3% soluble starch, corn steep
Liquor i%, raw soy flour 1%, polypeptone 0.5%, sodium chloride 0.3% and precipitated calcium carbonate 0.
500 mQ of seed culture medium consisting of 5% (pH 7.0)
After dispensing into a Qyosaka flask and incubating at 1200C for 20 minutes, Streptomyces sp. S-4562
8 stocks (IF○14980, FERM BP-2718
) and cultured on a reciprocating shaker for 40 hours at 28°C.

500mf2 of the seed culture thus obtained was mixed with 3ON of seed culture medium of the same composition as above (tap, antifoaming agent Actol 0).
．． 28°
Aeration and agitation culture for 42 hours at C [aeration rate 100% (30C/
minutes), stirred and rotated at 280 revolutions per minute.

The thus obtained seed culture 6Q was mixed with 05% glucose, 5% dextrin, 2% defatted soybean, 15% corn gluten meal, 1 lucium, 0.7% lucium, and 0.05% actochol (pH
7,0) main culture medium 120 (including 200Q,
Cultured with aeration and agitation for 66 hours at 28°C in a large tank [aeration rate ioo% (120M min), agitation.

It was rotated at 150 revolutions per minute to obtain culture 95Q.

Example 2 The culture solution (95Q) obtained in Example 1 was filtered using Hyfloth-Basel (Johns-Manville, USA) as a filter aid. Methanol (1
0012), stirred at room temperature for 1 hour, and
Filter the obtained extraction filter using Barcel, approximately 2'O
After concentrating to Q, the pH was adjusted to 70 and ethyl acetate (10Q
) was extracted twice.

The ethyl acetate layer (18Q) was washed with water (1012) and then concentrated to obtain a crude powder (43, 59). This crude powder was subjected to column chromatography on silica gel (5009, Merck & Co., West Germany), and a chloroform-methanol mixed solvent (30:1.3.0 (1, rr, 1-6.201.
3.0(, fr, 7-12; ] O: 1.3.0Q
, fr, 13-18; 5:I, 3.0Q, fr,
192 24). Fraction No. 6 contained compound TAN-1323A; fraction No. 7 to 11 contained T
AN-1323B was eluted.

Fraction Nos. 17 to 24 were collected and concentrated to obtain crude crystals of TAN-1323D (158 g), which were roughly recrystallized from methanol-water to give TA
Colorless scaly crystals (915xy) of N-1323D were obtained.

Fraction Nos. 13 to 15 were collected and concentrated to obtain a powder (1,480) containing TAN-1323C. This was subjected to silica gel (509) column chromatography and chloroform-methanol (30:1. 3
00+(!, fr, 1-6: 20:1.600mQ,
rr,? -], 8), fraction No. 16 to 18 were collected and concentrated, and crystallized from methanol-water to give TANl 323C (1
41m9: was obtained as colorless scaly crystals.

Effect of the invention Physiologically active substances TAN-1323A and B of the present invention.

C and D have an angiogenesis inhibitory effect and are useful for the prevention and treatment of various diseases accompanied by abnormal proliferation of angiogenesis, especially tumors.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of TAN-1323C, Figure 2 shows the 1HNMR spectrum of TAN-1323C, Figure 3 shows the infrared absorption spectrum of TAN-1323D, and Figure 4 shows the 'HNMR spectrum of TANl, 323D. are shown respectively.

Claims (3)

[Claims] (1) Physiologically active substance TAN-1323 represented by the following formula
C or D or a salt thereof. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R indicates a group represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼. ] (2) Belongs to the genus Streptomyces and is a physiologically active substance TAN
-1323C and/or D is cultured in a medium, and the physiologically active substance TAN is added to the culture.
- Physiologically active substance TAN-1 characterized by producing and accumulating 1323C and/or D and collecting the same.
323C and/or D manufacturing method. (3) An angiogenesis inhibitor containing the physiologically active substance TAN-1323A, B, C and/or D.